Pyruvate flux distribution in NADH-oxidase-overproducing Lactococcus lactis strain as a function of culture conditions

Abstract

The influence of growth conditions on product formation from glucose by Lactococcus lactis strain NZ9800 engineered for NADH-oxidase overproduction was examined. In aerobic batch cultures, a large production of acetoin and diacetyl was found at acidic pH under pH-unregulated conditions. However, pyruvate flux was mainly driven towards lactate production when these cells were grown under strictly pH-controlled conditions. A decreased NADH-oxidase overproduction accompanied the homolactic fermentation, suggesting that the cellular energy was used with preference to maintain cellular homeostasis rather than for NADH-oxidase overproduction. The end product formation and NADH-oxidase activity were also studied in cells grown in aerobic continuous cultures under acidic conditions. A homoacetic type of fermentation as well as a low NADHoxidase overproduction were observed at low dilution rates. NADH-oxidase was efficiently overproduced as the dilution rate was increased and consequently metabolic flux through lactate dehydrogenase drastically decreased. Under these conditions the flux limitation via pyruvate dehydrogenase was relieved and this enzymatic complex accommodated most of the pyruvate flux. Pyruvate was also significantly converted to acetoin and diacetyl via K-acetolactate synthase. At higher dilution rates, acetate production declined and the cultures turned to mixed-acid fermentation. These results suggest that the need to maintain the cellular homeostasis influenced NADH-oxidase overproduction and consequently the end product formation from glucose in these engineered strains. ß

Effects of Cultivation Conditions on Folate Production by Lactic Acid Bacteria

“…We assume that hemin stimulates the direct oxidation of NADH by oxygen. As a consequence, NADH levels decrease and pyruvate is consumed via alternative nonreducing pathways, resulting in decreased acidification (less lactate, more acetoin) (18). Recently, it was reported that during aerobic growth the addition of hemin extends the growth period (10) and that hemin may reconstitute proton extrusion (5).…”

IS 981 -Mediated Adaptive Evolution Recovers Lactate Production by ldhB Transcription Activation in a Lactate Dehydrogenase-Deficient Strain of Lactococcus lactis

“…The las operon-encoded lactococcal Ldh protein converts pyruvate to lactate with high efficiency. Moreover, through the concomitant conversion of NADH to NAD ϩ , this reaction provides the electron sink required to maintain redox balance, which has been shown to be a critical determinant in the control of pyruvate flux in L. lactis (9,18,35,41).Construction of defined ldh disruption mutants of L. lactis has allowed redistribution of the lactococcal pyruvate pool toward products other than lactate (20,23,26,35,43,50). Under aerobic conditions, the ldh-deficient strains displayed an almost complete loss of lactate production and acetoin was found to be the main end product of fermentation, while the amounts of other metabolic end products like acetate, butanediol, ethanol, and formate appeared to depend on the fermentation conditions applied (23,43).…”

“…The las operon-encoded lactococcal Ldh protein converts pyruvate to lactate with high efficiency. Moreover, through the concomitant conversion of NADH to NAD ϩ , this reaction provides the electron sink required to maintain redox balance, which has been shown to be a critical determinant in the control of pyruvate flux in L. lactis (9,18,35,41).…”

“…Construction of defined ldh disruption mutants of L. lactis has allowed redistribution of the lactococcal pyruvate pool toward products other than lactate (20,23,26,35,43,50). Under aerobic conditions, the ldh-deficient strains displayed an almost complete loss of lactate production and acetoin was found to be the main end product of fermentation, while the amounts of other metabolic end products like acetate, butanediol, ethanol, and formate appeared to depend on the fermentation conditions applied (23,43).…”

“…Under aerobic conditions, the ldh-deficient strains displayed an almost complete loss of lactate production and acetoin was found to be the main end product of fermentation, while the amounts of other metabolic end products like acetate, butanediol, ethanol, and formate appeared to depend on the fermentation conditions applied (23,43). The presence of molecular oxygen allows the cells to maintain their redox balance through the activity of the endogenous NADH oxidase, thereby sustaining rapid sugar fermentation by the ldh-deficient cells under aerobic conditions (23,35). However, under anaerobic conditions, the rate of sugar fermentation is reduced in these cells and the main metabolic end products observed were formate, ethanol, and butanediol (43).…”